We have used large-insert cloning to obtain yeast and bacterial artificial chromosome clones (YACs and BACs) containing two genes of importance in human reproduction: PLAC1, expressed uniquely in one of the three layers of the placenta, and FOXL2, expressed only in developing eyelids and in follicular cells of the ovarian follicles. [Deficiency in FOXL2 leads to Premature Ovarian Failure (POF) in some women.] The goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. In contrast to other instances such as liver-specific genes, the tissue specificity is not reproduced by segments of putative promoter DNA up to 10 Kb 5? of PLAC1 or FOXL2. This indicates that longer-range regulation is operative. Consistent with that notion, a translocation that disrupts transcription of FOXL2 lies very far (about 168 Kb away) from the gene sequence. In the next phase of these studies we are focusing on both human and mouse Plac1 genes, Using recombination-based methods in bacteria, we are exploring the incorporation of GFP (green fluorescence protein) as a reporter gene into a BAC containing the human PLAC1 gene, along with a drug marker selectable in mammalian cells. In preliminary studies, a BAC containing the human PLAC1 gene with at least 60 Kb of upstream sequences, retrofitted with a GFP cassette in the 3'UTR region, gives expression in Jeg3 choriocarcinoma cell line, again consistent with long-range regulation. The aim now will be to resect the promoter region systematically in derivatives of the BAC, and use transfection studies to define the upstream regulatory regions in DNA and features of the corresponding chromatin structure. In a further approach at the protein level, experiments with rabbit antibodies raised against selected epitopes from both human and mouse proteins suggest that the PLAC1 protein exists in vivo as a dimer, undergoes substantial protein modifications, and is localized in the cytoplasm. Experiments are in progress to follow the human PLAC1 protein with the FLAG epitope, and to look for interacting partners by immunoprecipitation with anti-FLAG antibodies. The FLAG segment distinguishes the BAC-encoded PLAC1 from endogenously expressed protein. In conjunction with these studies, the mouse Plac1 gene is also being fused with GFP to construct a transgene and determine its expression pattern during development, in collaboration with Dr. M. Fant, who is generating a mouse knockout. Based on microarray data, we have also recently identified two more genes, Plac8 and Plac9, whose expression is enriched in placenta. They provide further targets for eventual comparative analysis of regulatory signals; and comparable approaches are also projected for the FOXL2 gene after the PLAC1 model is characterized.